I. Field of the Invention
The present invention is directed to methods and compositions for prophylaxis and treatment of cell membrane damage, and resultant tissue injury, caused by external sources capable of disrupting cell membrane integrity. In particular embodiments, the present invention relates to methods of prophylaxis and/or treatment of tissue damage at the cell membrane level caused by radiation, freezing, or certain types of mechanical forces, and to the enhancement of cell survival and transfection efficacy, through the sealing of permeabilized cell membranes with an effective amount of a composition comprising a surface-active copolymer, preferably through pretreatment or concomitant treatment of injured tissue or permeabilized and transfected cells with high energy phosphate compounds.
II. Background of the Invention
The widespread clinical use of radiation therapy in the treatment of cancer, and the increasing interest in medicine and the biological sciences in methods involving the insertion of foreign molecules into living cells, have surprisingly led to a common problem which the present invention addresses. That problem involves damage to the membranes of cells in the nature of membrane permeabilization, or the production of discrete openings at numerous sites in the cells' membranes. The consequences of membrane permeabilization are numerous, and include loss of cytoplasm and some of the contents thereof, disruption of ionic concentration gradients, and depletion of intracellular energy stores.
In radiation therapy cells in normal tissue within the radiation field often suffer cell membrane permeabilization. The permeabilized cells may thereafter die and the tissue will subsequently undergo necrosis. In the laboratory, the methodologies typically used to insert foreign molecules into cells often involve the deliberate permeabilization of the cells' membranes, the openings serving as a site of ingress for the foreign molecules. As noted, however, one of the consequences of the permeabilization is concomitant egress of the contents of the cell, and without some means of potentiating the repair of the openings, cell survival rates can often be unacceptably low.
One of the more serious consequences of cell membrane permeabilization is the significant depletion of intracellular energy stores. Under normal circumstances, cells maintain a high level of ATP by using oxidizable substrates as sources of free energy. Following permeabilization, a great deal of energy would be expended in the cells' attempts to maintain intracellular ionic balances as ions move through openings in the cell membrane down their concentration gradients. As the intracellular ionic environment is disrupted, the normally occurring intracellular reactions which regenerate ATP stores will be inhibited, and as energy-dependent cellular processes continue, both to maintain cellular function and to attempt to repair the damage to the cell, the cells' ATP stores will be depleted further. This can result in the cells' inability to synthesize macromolecules necessary for continued cellular function, their inability to re-establish the proper ionic gradients across the membrane, and finally cell death.
A system in which such a depletion of ATP has been well demonstrated is in animals suffering hemorrhagic shock. It has been demonstrated that administration of ATP-MgCl.sub.2 before, during, and even after a period of severe shock in rats had a beneficial effect on the animals' survival. The ATP was administered along with the MgCl.sub.2 in order to prevent chelation of divalent cations from the vascular system by ATP administered alone. Furthermore, MgCl.sub.2 inhibits the deamination and dephosphorylation of ATP. Thus, by administering equimolar amounts of ATP and MgCl.sub.2, a higher concentration of ATP will be available to the tissues than if the ATP were administered alone. The results of this study suggested that the beneficial action of ATP-MgCl.sub.2 may not have been through vasodilation alone, and it was postulated that the administered ATP could have a "priming effect" on the intracellular synthesis of ATP (1).
The actual method of cell membrane repair in vivo remains unknown, although researchers have made some inroads toward understanding the mechanisms involved. Calcium ions have been implicated, through both in vitro and in vivo studies, as having a critical role in membrane fusion and repair (2,3,4). Membrane and cytoskeletal proteins, including spectrin, dystrophin, and actin are probably also actively involved in the maintenance and repair of the cell membrane in vivo (4). Finally, it has also been suggested that chemical factors may play a signal-like role in wound healing at the cellular level (4). The present invention provides a method for minimizing radiation-induced damage via prophylaxis or via potentiation of cell membrane repair by post-exposure treatment with a surface active copolymer, as well as a method for potentiating membrane repair and transfection efficiency in vitro.